The continuing growth of the global population is driving an increased demand for food resources and raw materials for food processing. According to United Nations projections, the world population is expected to reach approximately 10 billion by 2050. To mitigate the risk of food shortages and to strengthen food security, many countries are seeking new strategies that promote environmentally sustainable crop production technologies and the advancement of related industries. Globally, one of the major obstacles to crop yield improvement is the annual loss attributed to pests and diseases, which is estimated to cause economic damage of approximately USD 22 billion (Savary et al., 2019). In addition to biotic factors such as plant viruses, pathogens, and insect pests, global climate change and variability have a profound effect on the emergence and spread of crop diseases, including viral infections. These factors also influence plant physiological responses, pollinator dynamics, pollination efficiency, and fruit development, thereby affecting agricultural productivity through multiple pathways (Singh et al., 2023). Although numerous approaches have been developed for the management of plant viral diseases, no effective pesticides or therapeutic agents have yet been commercialized for plant viruses. Consequently, early diagnosis and the rapid removal of infected plants remain among the most practical strategies currently implemented in the field.
Historically, Sanford and Johnston proposed the concept of pathogen-derived resistance in 1985 (Sanford and Johnston, 1985). Based on this concept, numerous attempts were made to express viral genes or genome fragments in transgenic plants to confer virus resistance, and several of these efforts proved successful (Baulcombe, 1994; Baulcombe, 1996; Beachy, 1993; Lomonossoff, 1995; Wilson, 1993). Many approaches extending this concept achieved practical outcomes, and some resulted in the development of virus-resistant cultivars of potato and squash for commercial application. Among the most notable early successes was the demonstration in 1987 that expression of the tobacco mosaic virus (TMV) coat protein (CP) gene in tobacco conferred resistance to TMV, thereby establishing that proteins such as replicases, CPs, movement proteins, and antiviral peptides could artificially mediate virus resistance in plants (Baulcombe, 1996). By the mid-1990s, however, a natural regulatory mechanism known as RNA interference (RNAi), or post-transcriptional gene silencing (PTGS), had been discovered in plants (Baulcombe 2004; Lindbo and Dougherty, 1992; Napoli et al., 1990). RNAi was subsequently identified in Caenorhabditis elegans in 1998 as the mechanism underlying PTGS (Fire et al., 1998), and subsequent studies demonstrated that double-stranded RNA (dsRNA) could trigger RNAi in fungi and mammals as well (Elbashir et al., 2001; Miller et al., 2012). Since viral RNAs have been established as common inducers of RNA silencing pathways in plants, leading to resistance against viral infection (Lindbo and Dougherty, 1992; Voinnet et al., 1999). In this process, Dicer-like proteins process endogenous or exogenously introduced dsRNA into 21-24 nt small interfering RNA (siRNA) duplexes. These duplexes associate with RNA-binding proteins such as DRB4, which in turn interact with RNA-dependent RNA polymerases (RDR1, RDR6). Amplification by plant RdRPs generates secondary siRNA duplexes, which are loaded onto ARGONAUTE proteins; the passenger strand is degraded, and the guide strand directs formation of the RNA-induced silencing complex (RISC) (Baulcombe, 2004; Fig. 1). The RISC complex binds complementary transcripts and cleaves them, thereby downregulating gene expression.
RNAi technologies have largely been implemented through genetic modification to induce gene silencing within plants. However, many crop species remain recalcitrant to transformation due to the lack of efficient regeneration protocols following tissue culture, the high costs of inputs, and extended time requirements.
In addition, public perception of genetically modified organisms (GMOs) remains cautious (James, 2010), and growers have expressed concerns regarding incomplete assurances of biosafety in commercial cultivation. As an alternative non-GM strategy for managing viral diseases and other pests, there has been increasing attention. Among these, spray-induced gene silencing (SIGS), which delivers externally produced dsRNA molecules to plants via foliar sprays, seed treatments, trunk injections, or uptake through cuttings and roots, has emerged as a promising approach (He et al., 2024; Hoang et al., 2022; Tenllado and Díaz-Ruíz, 2001; Uslu and Wassenegger, 2020). SIGS offers greater flexibility for experimental design and functional analysis than transgenic overexpression or knockdown, requires only RNA-based inputs, and is perceived as environmentally safer, thereby representing a sustainable plant protection technology (Vatanparast et al., 2024). Importantly, SIGS is designed to affect only target viruses while leaving plants unaffected, with the added advantage of minimizing off-target effects (Neumeier and Meister, 2021). For SIGS to be successfully commercialized, several requirements must be addressed: (i) scalable production platforms that reduce costs and labor inputs (Aalto et al., 2007), (ii) formulation technologies that ensure efficient and user-friendly application in the field, and (iii) comprehensive validation of environmental biosafety, including non-target organisms such as pollinators and soil microbes (Hoang et al., 2022; Uslu and Wassenegger, 2020; Parker et al., 2019).
In this review, we not only summarize recent advances in the development and application of dsRNA-based SIGS technologies, but also distinguish our perspective from previous reviews by integrating progress in delivery systems with a critical analysis of their practical effectiveness, safety, and scalability in plants, particularly on practical field application (Islam et al., 2025). We further provide an up-to-date assessment of industrial-scale RNA production, emphasizing how recent advances in in vitro synthesis, microbial fermentation and bioreactor-based platforms have reduced production costs to levels compatible with large-scale agriculture (Aalto et al., 2007; Bally et al., 2018), thereby uniquely strengthening the case for realistic field deployment of dsRNA sprays in greenhouse and open environments (Mitter et al., 2023; Niehl et al., 2018).
SIGS: Principles of Exogenous dsRNA Delivery
As outlined above (Fig. 1), the intrinsic RNAi machinery in plants has been exploited through exogenous RNA interference (exo-RNAi), whereby externally derived RNAi triggers are applied to enhance resistance against plant viruses. Building on this theoretical framework, SIGS was developed. SIGS involves the direct application of externally produced dsRNA to crops, representing one of the most widely investigated strategies for agricultural control of viruses, fungal pathogens, and insect pests (Bolognesi et al., 2012; Kumar et al., 2025; Li et al., 2015; Zhang et al., 2015). Introduction of target-specific dsRNA into an appropriate host plant through a transgenic expression system can confer effective protection against plant pathogen infection. As first proposed two decades ago, this biotechnology approach known as host-induced gene silencing (HIGS) was considered as a promising alternative to conventional plant protection strategies because of its high specificity to genes of target pathogens. Moreover, HIGS exhibits minimal environmental and toxicological side effects compared with protein-producing transgenes or chemical control measures (Koch and Kogel, 2014; Zeng et al., 2019). Over the past decade, numerous studies have demonstrated the utility of HIGS for controlling fungal diseases, with compelling evidence reported in landmark studies published in 2010 and thereafter (Nowara et al., 2010). For example, expression of a hairpin interference cassette targeting the GUS reporter gene (hpGUS) in tobacco plants suppressed expression of this gene in Fusarium verticillioides. A clear HIGS-mediated protective effect was also observed in cereals infected with the powdery mildew pathogen Blumeria graminis (Nowara et al., 2010). In contrast to fungal pathogens, HIGS against plant viruses directly elicits intracellular silencing effects that can be readily quantified (i.e., target virus titer, development of symptom induction), and thus has been widely accepted as a promising alternative antiviral strategy. Notably, transgenic expression of dsRNA using the NIa protease of potato virus Y (PVY) with a hairpin intron system in tobacco plants has achieved complete suppression of PVY infection (Wesley et al., 2001).
In most cases in SIGS application, dsRNA is delivered by foliar spraying, where molecules applied to the leaf surface can be locally absorbed. Importantly, subsequent systemic movement of dsRNA has been reported, conferring whole-plant protection and thus providing acquired resistance against diverse viral and pathogenic challenges (Dalakouras et al., 2024; Uslu et al., 2020). From a preventive perspective, SIGS may function analogously to RNA vaccines in animals, as it enables rapid and sequence-specific induction of resistance. By selecting viral gene regions that effectively trigger RNAi while minimizing off-target effects on host transcripts (Chen et al., 2021), SIGS can confer targeted protection within a short time frame. This approach is particularly advantageous for crop species lacking natural virus resistance sources or where resistance genes are present only in distantly related species, thus limiting introgression by conventional breeding. Moreover, in the case of newly introduced or emerging viruses that threaten agricultural production, SIGS provides a practical option for inducing resistance without altering the host genome (Voloudakis et al., 2022). It is noteworthy that exogenous dsRNA can serve as a bifunctional molecule in plants, activating sequence-specific RNAi and a pattern-triggered immunity (PTI)-like response. Spraying potatoes with PVY-specific dsRNA led to robust RNAi, showing accumulation of PVY-derived siRNAs and concomitant induction of PTI marker transcripts, such as WRKY29, EDS5, and PR-1b (Samarskaya et al., 2022). Functionally, the treatment selectively suppressed PVY, whereas infection by the unrelated PVX was unchanged despite PTI activation in PVX-challenged tissues (Samarskaya et al., 2022). These data indicate that, under our conditions, homolog-directed RNAi is the proximate determinant of protection, while PTI alone is insufficient to curb PVX (Samarskaya et al., 2022). Therefore, exogenous dsRNAs can act as multi-tools that mainly trigger RNAi as the key mechanism of antiviral resistance, as well as induce other mechanisms involved in PTI (Samarskaya et al., 2023; Venu et al., 2024). Applications of SIGS across multiple crop-virus systems, highlighting target genes and host plants, are summarized in Table 1. Moreover, recent studies have demonstrated that exogenously applied dsRNA can remain biologically active for several days under controlled conditions, supporting the concept that SIGS may provide short-term, vaccine-like protection against rapidly spreading viral outbreaks (Bachman et al., 2020; Voloudakis et al., 2022).
Constraints of SIGS Technology
Limitations in dsRNA stability and durability
The persistence and stability of exogenously applied dsRNA on the leaf surface are critical determinants of RNAi efficacy. Prior to cellular uptake, dsRNA should overcome multiple physical and chemical barriers that plants have evolved against a broad range of biotic and abiotic stresses. Because plant viruses complete their replication, movement, and assembly exclusively inside host cells, exogenous dsRNA must also enter the cell to induce antiviral RNAi responses or resistance (Fig. 1). From this perspective, locally sprayed dsRNA under SIGS conditions should remain on the leaf surface long enough to act as a continuous source for uptake, since immediate intracellular entry is limited.
The persistence of sprayed dsRNA is strongly influenced by environmental factors such as ultraviolet (UV) radiation, temperature, and pH, all of which can accelerate degradation and thereby reduce RNAi efficacy. For instance, exposure to UV for approximately one hour has been shown to degrade dsRNA and abolish its biological activity (San Miguel and Scott, 2016). The extent to which UV exposure under field or greenhouse conditions affects RNA stability and SIGS efficacy remains insufficiently understood and requires further study. Surface pH also influences dsRNA persistence; RNA is chemically more stable under acidic than alkaline conditions. Some studies indicate that dsRNA exhibits limited resistance to alkaline hydrolysis, but stabilization strategies, such as incorporating protective carriers, are necessary to enhance persistence (Hegg et al., 1997; Parker et al., 2019). Field monitoring has revealed that rainfall exceeding 20 mm within 24 h can remove a significant proportion of foliar dsRNA deposits, highlighting the urgent need for rain-fast formulations to maintain antiviral efficacy (Bachman et al., 2020).
Limitations in cellular penetration of dsRNA
The effectiveness of SIGS is dependent on the sufficient uptake of dsRNA, which is itself influenced by environmental conditions such as irrigation or rainfall that alter water retention on leaf surfaces (Fig. 2). Key determinants of leaf absorptivity include physical and chemical features such as the cuticle, cuticular waxes, and trichomes (Brewer et al., 1991; Burgess and Dawson, 2004; Eller et al., 2013; Martin and von Willert, 2000). A high density of trichomes increases surface roughness and hydrophobicity, reducing wettability and consequently limiting the absorption of aqueous formulations, including dsRNA sprays (Khayet and Fernández, 2012). Because dsRNA molecules are generally applied in hydrophilic aqueous solutions, leaf wettability critically affects droplet adhesion, surface retention, and subsequent penetration. On leaves with low wettability (greater hydrophobicity), sprayed dsRNA tends to bead and roll off, resulting in reduced uptake. Cuticular permeability is enhanced under elevated temperature and for smaller solutes, and while plasmodesmata are generally considered confined to internal tissues, related structures termed ectodesmata have been described in epidermal cell walls (Franke, 1961). Although direct diffusion through cuticular pores or ectodesmata has been hypothesized, the accessibility of these structures beneath the cuticle suggests they contribute minimally to dsRNA absorption (Rundel, 1982). To date, the primary pathway implicated in foliar uptake of dsRNA during SIGS is stomatal entry (Kiselev et al., 2021). Uptake through stomatal apertures and guard cells appears to be the most plausible route. Once internalized, the degree of dsRNA uptake by mesophyll cells is likely modulated by tissue hydrophilicity, the presence of phenolic compounds, polysaccharides, mucilage cells, and gradients in water potential (Aparecido et al., 2017; Ghosh et al., 2018; Schönherr and Bukovac, 1972). In addition, differences in cuticular structure among crop species suggest that uptake efficiency is host-dependent, necessitating species-specific optimization of SIGS delivery systems (Niehl et al., 2018).
Strategies to Overcome the Limitations of SIGS
Direct dsRNA delivery systems
Due to the anatomical and physicochemical characteristics of stomata, the uptake of foliar-applied dsRNA into internal leaf tissues is inherently constrained. To enhance RNAi efficacy, strategies have been developed to increase leaf wettability, promote cuticular penetration, and facilitate solute transport into plant cells. Physical approaches include simple spraying, syringe-based infiltration, abrasion to disrupt the cuticle, and high-pressure spraying for forced delivery of dsRNA (Burkhardt et al., 2012; Dalakouras et al., 2016; Eichert et al., 2008; Huang et al., 2020). Conflicting findings have been reported regarding high-pressure spraying. In one study, transgenic Nicotiana benthamiana expressing GFP (called 16c), which did not exhibit silencing following standard spraying or infiltration, displayed both local and systemic GFP silencing when treated with GFP-derived siRNA by high-pressure spraying (Dalakouras et al., 2016). In contrast, a subsequent study from the same group using the identical transgenic line found no GFP silencing despite high-pressure dsRNA application (Uslu et al., 2020; Uslu et al., 2022). The authors attributed these discrepancies to insufficient dsRNA uptake by plant cells, leading to inadequate amplification of secondary siRNAs by the host RNA silencing machinery. These contrasting results emphasize the need to systematically compare different direct delivery methods (e.g., Agrobacterium-mediated dsRNA expression, siRNA infiltration) and to optimize conditions for effective dsRNA entry. Moreover, for antiviral applications, it is essential to evaluate both long dsRNAs and synthetic virus-specific siRNAs, as dsRNA length and molecular form strongly influence silencing efficacy. Timing of dsRNA application relative to virus infection and the durability of induced resistance are also critical for successful SIGS application to crops. It should be considered that virus-infected susceptible plants generate abundant virus-derived siRNAs but still permit virus replication, movement, and symptom development, suggesting siRNA generation is not a prerequisite for showing resistance to plant viruses in crops.
Chemical methods offer additional opportunities for enhancing dsRNA uptake. Representative strategies include the use of surfactants and chemical regulation of stomatal aperture. Surfactants, widely used in combination with chemical pesticides for crop protection, reduce surface tension, improve droplet spreading, and thereby increase penetration. For example, application of the surfactant Silwet L-77, combined with low-pressure spraying (2.5 bar) of a 685 bp dsRNA or a synthetic 21-nt siRNA, successfully induced silencing of the phytoene desaturase gene in N. benthamiana (Sammons et al., 2011; Sanan-Mishra et al., 2021). Surfactant-mediated delivery is currently regarded as one of the most practical approaches for enabling large-scale dsRNA entry into plant cells, and several studies have continued to investigate its utility (Castro et al., 2013; Dalakouras et al., 2024; Tenllado et al., 2003; Whitfield et al., 2018).
The encapsulation technology of dsRNA has been developed to enhance dsRNA yield in microbial hosts and to protect it from degradation under environmental conditions. A platform named AgriCell technology (AgroSpheres, www.agrospheres.com) was developed to enable simultaneous production and encapsulation of dsRNA. This technology employs a non-GMO approach to generate bioparticles that encapsulate dsRNA produced during bacterial fermentation. The encapsulation protects dsRNA from physical and chemical degradation factors such as heat, ultraviolet radiation, and microbial nucleases, ensuring efficient delivery and stable release (Hough et al., 2022). Using this system, dsRNA can be produced at yields of approximately 100 mg per liter, and it has been reported to suppress gray mold disease in strawberries (Islam et al., 2021). However, there are currently no reports demonstrating antiviral efficacy of virus-specific dsRNA introduced into plants via the AgriCell approach. Nonetheless, this method shows strong potential for future applications due to the enhanced physical and chemical stability conferred by the encapsulated particle structure, equivalent to nanoparticle-based delivery systems.
Nanomaterial-based dsRNA delivery systems
To overcome multiple physical and biochemical barriers and to enhance the protection and efficiency of topical dsRNA delivery in plants, nanoparticles ranging from 1 to 500 nm have been developed. In general, formulated dsRNA exhibits greater stability, cellular uptake, and overall efficiency compared with non-formulated dsRNA (Hamid and Saleem, 2022; Mitter et al., 2017; Mitter et al., 2023). Because dsRNA is negatively charged, positively charged nanoparticles, including metals or cationic polymers, are designed to electrostatically interact with the phosphate backbone of dsRNA, thereby stabilizing the dsRNA formulation (Fig. 3). These interactions generate dsRNA-nanoparticle complexes that carry an overall positive charge, facilitating interaction with the negatively charged plasma membrane (Dutta et al., 2022). In addition to conferring protection against environmental degradation, such as UV radiation and microbial nucleases, nanomaterials have been shown to form biodegradable complexes that allow sustained dsRNA release over time (Dutta et al., 2022; Ma et al., 2022). Among the most promising formulations for commercialization of dsRNA-based antivirals are layered double hydroxide (LDH) nanoclays, which have demonstrated efficacy against multiple plant viruses (Mitter et al., 2017). LDH nanoclays loaded with dsRNA, commonly referred to as “BioClay” gradually disintegrate upon exposure to atmospheric CO2 and moisture, releasing dsRNA that is subsequently absorbed into plant cells, likely by passive diffusion. This system confers protection of dsRNA from degradation and wash-off for at least 30 days (Hernández-Soto and Chacón-Cerdas, 2021; Mitter et al., 2017; Niño-Sánchez et al., 2022; Qiao et al., 2021). Beyond viral targets, LDH-based formulations have also been applied against plant genes and fungi (Niño-Sánchez et al., 2022) as well as for the control of Bemisia tabaci in potato plants (Jain et al., 2022). Successful applications include suppression of bean yellow mosaic virus and cucumber mosaic virus replication using LDH-based carriers (Elbeshehy et al., 2015; El-Sawy et al., 2017). Additionally, BioClay-based dsRNA has successfully inhibited aphid-mediated transmission of BCMV, while carbon dots and chitosan have enhanced dsRNA absorption and longevity (Havrdová et al., 2016; Mitter et al., 2017; Song et al., 2018). Chitosan (poly β-1,4-D-glucosamine) is another example of interest. At pH <6, protonated amine groups in chitosan interact with the anionic phosphate backbone of dsRNA to form chitosan/dsRNA polyplex nanoparticles (PNs). Recent studies have demonstrated that these PNs upregulate expression of the clathrin heavy chain gene, thereby activating the clathrin-dependent endocytosis pathway and ultimately improving dsRNA stability, cellular uptake, and RNAi efficiency (Zhou et al., 2023). Similarly, carbon dots have been employed for siRNA delivery, resulting in enhanced cellular uptake under low-pressure spraying and inducing silencing of endogenous plant genes (Das et al., 2015). Single-walled carbon nanotubes (SWNTs) have also been investigated as nanocarriers for delivering DNA and siRNAs into intact plant cells. Cellular uptake of SWNT/DNA conjugates revealed the potential of SWNTs to function as nano-transporters targeting diverse organelles (Demirer et al., 2019). More recently, SWNTs have been shown to protect siRNAs from nuclease degradation and to efficiently deliver DNA and siRNAs into the cytoplasm, thereby inducing gene knockdown (Zhang et al., 2022).
Nanocarriers also include virus-like particles (VLPs), which encapsulate dsRNA into proteinaceous structures that protect against nucleolytic degradation and facilitate delivery of target dsRNAs to host cells, effectively triggering post-transcriptional gene silencing against pathogens (Hamid and Saleem, 2022; Xue et al., 2023). For example, Apse RNA containers utilize MS2 bacteriophage CP to produce self-assembling VLPs. These VLPs are encapsulated during bacterial growth in E. coli, offering high stability and efficient delivery (Dalakouras et al., 2024; Zotti et al., 2018). Lipid-based vesicles or liposomes contained inside with gold nanoparticles, copper nanoparticles, guanidine-based polymers, and surfactant formulations have also been explored to accelerate cellular uptake and promote RNAi induction upon foliar spraying (Hegg et al., 1997; Mathur et al., 2025; Zhu and Palli, 2020). Although research on nanocarrier-mediated dsRNA delivery remains limited and highly efficient delivery systems are still under development, current evidence indicates that further advances in nanodelivery technologies will be essential for translating topical RNAi sprays into practical field applications (He et al., 2022; Hernández-Soto and Chacón-Cerdas, 2021; Menezes et al., 2022). However, nanoparticle-RNA complexes may also interact with soil microbiomes, with preliminary evidence showing shifts in microbial diversity and nutrient cycling efficiency, underscoring the need for comprehensive environmental risk assessment (Luo et al., 2024; Parker et al., 2019).
Cost-effective dsRNA production
Currently, large-scale dsRNA production can be achieved by two main approaches: in vivo systems using genetically engineered microorganisms and in vitro synthesis using RNA polymerase. As an early proof-of-concept method, the RNase III-deficient Escherichia coli strain HT115(DE3) together with the pL4440 plasmid has been widely employed for dsRNA production under isopropyl β-D-1-thiogalactopyranoside (IPTG) induction (Timmons and Fire, 1998). The initial expression plasmid contains a pair of oppositely oriented T7 promoters flanking the target sequence but lacks transcriptional terminators, often resulting in nonspecific RNA transcription. To overcome this limitation, strategies incorporating and optimizing transcriptional terminators have been developed to improve dsRNA yield (Sturm et al., 2018; Ross et al., 2024). Beyond strain and plasmid engineering, optimization of culture media composition, fermentation conditions, and inducers (e.g., replacement IPTG with skim milk) has been shown to enhance dsRNA production efficiency (Hough et al., 2022; da Rosa et al., 2024). In addition to E. coli, other biosafety strains such as Saccharomyces cerevisiae, Pseudomonas syringae and Bacillus subtilis have been explored as hosts for dsRNA expression (Hough et al., 2022). However, because the use, distribution, and application of these recombinant strains in agricultural settings are classified as GMOs in many countries, regulatory approval is required before commercialization or field application (Vatanparast et al., 2024). An alternative approach to circumvent GMO-related issues is in vitro dsRNA synthesis. Notably, GreenLight Biosciences (2017) has developed a cell-free production platform that utilizes cellular RNA to generate nucleotide triphosphates (NTPs), the key substrates for in vitro dsRNA synthesis, thereby significantly reducing production costs (GreenLight Biosciences, 2024). Using this system, the production cost can be lower than $1 per gram of dsRNA, making large-scale dsRNA synthesis economically feasible.
Field application
Although we failed to extract meaningful statistics of SIGS only from some databases, the number of patent applications related to RNAi-based crop protection steadily increased from 5 in 2003 to 392 in 2011 and remained relatively high until 2016. Between 2003 and 2023, a total of 275 patents related to RNAi-based crop protection were reported to be registered 2021 (Germing et al., 2025). Meanwhile, the number of research publications on RNAi-based crop protection increased continuously from 2013 to 2016 and showed a gradual annual rise from 2016 to 2021 (Germing et al., 2025). This trend indicates that RNAi technologies, including SIGS, have progressed beyond the conceptual and experimental validation stages to the phase of technology transfer and commercialization within industry.
To effectively control target viruses or plant pathogens (i.e., fungi) using dsRNA, the development process can be divided into three major stages. Stage 1 is “concept design and validation”. This stage involves the selection, design, and functional verification of precise target genes that are essential for viral replication or pathogenicity. Bioinformatics tools via artificial intelligence can be used to identify highly conserved genomic regions involved in viral replication and movement. As summarized in Table 1, the most common targets include the RNA-dependent RNA polymerase (RdRp) gene, viral silencing suppressor (VSR) gene, and CP gene. Combining multiple gene targets in a single construct can compensate for low effects associated with individual targets. Furthermore, designing dsRNA with self-splicing intron sequences to form a hairpin structure can enhance antiviral efficacy within plant cells. Next, stage 2 is “dsRNA formulation and efficient delivery to plants”. This stage focuses on stabilizing dsRNA formulations and developing efficient delivery systems for plant uptake, particularly under unfavorable environmental conditions. In the case of SIGS, the antiviral protection of sprayed dsRNA typically lasts only a few days, requiring frequent reapplications, which this is an impractical approach for long-season field crops. With increasing labor costs and reduced agricultural workforce, repeated pesticide spraying has become economically and logistically challenging. Therefore, improving persistence, delivery efficiency, and stability of dsRNA formulations is a top research priority. Encapsulation of dsRNA using nanoparticles, virus-like particles (VLPs), or AgriCell technology can provide enhanced protection and controlled release. These approaches are likely to succeed first in high-value horticultural crops grown in controlled farm environments such as greenhouses or smart farms. In addition, developing technologies that enable root uptake and systemic movement of dsRNA throughout the plant represents an important future direction. This approach is particularly promising in hydroponic cultivation systems, where dsRNA can be efficiently absorbed through roots and applied easily across different crops. Stage 3 is “large-scale production, environmental safety, and commercialization”. At this final stage, dsRNA must be produced at industrial scale with long-lasting efficacy, no phyto-toxicity, and extended shelf life (several months to two years). Furthermore, it must be proven to be non-toxic, environmentally safe, and have no adverse effects on non-target organisms, including beneficial insects and humans. As a biopesticide, the production cost must remain low while maintaining field stability at ambient temperatures. Although SIGS has demonstrated strong efficacy under laboratory conditions, large-scale field trials remain limited and may involve significant trial and error. However, with the integration of advanced application technologies (such as drone-based precision spraying) it will become possible to achieve accurate delivery with minimal labor input, facilitating the practical and sustainable implementation of dsRNA-based biocontrol strategies (Mitter et al., 2023).
Regulatory approaches and safety of dsRNA for SIGS application
Current studies clearly demonstrate that the systemic absorption of dsRNA from RNAi-based pesticides in humans and vertebrates is highly improbable, a conclusion supported by the long history of safe dsRNA consumption through natural foods. Humans and livestock possess robust enzymatic degradation and cellular uptake barriers in the gastrointestinal tract, making any physiological effects from ingested dsRNA extremely unlikely (O’Neill et al., 2011).
Moreover, dsRNA generally exhibits low environmental stability in soil, sediments, and aquatic systems, indicating that it does not persist for extended periods (Bachman et al., 2020; Parker et al., 2019). Because formulation properties can influence both the environmental persistence of dsRNA and potential human exposure pathways, case-specific evaluations that consider formulation characteristics are essential during risk assessment (Bachman et al., 2020). Furthermore, since the formulations themselves may exert additional effects on non-target organisms or the environment, independent environmental impact assessments of the formulations should be conducted alongside the evaluation of dsRNA (De Schutter et al., 2022).
The regulatory frameworks for dsRNA-based pesticides vary across regions, with distinct differences among the United States, Australia, and the European Union (EU). In the United States, dsRNA-based products are classified as biochemical pesticides and require approval from the Environmental Protection Agency under the Federal Insecticide, Fungicide, and Rodenticide Act and the Federal Food, Drug, and Cosmetic Act (Dietz-Pfeilstetter et al., 2021; Leahy et al., 2014). In Australia, these products are regulated as agricultural chemical products under the Australian Pesticides and Veterinary Medicines Authority, while the Office of the Gene Technology Regulator considers SIGS as non-GMO under specific conditions dsRNA (De Schutter et al., 2022). In South Korea, however, there are currently no formal regulatory guidelines defining the safety evaluation or registration requirements for dsRNA-based chemicals as pesticides. The establishment of such a regulatory framework is expected to take time. The classification of dsRNA as either a chemical or biological pesticide remains ambiguous, highlighting the urgent need for legal and institutional clarification. It is critical to develop evaluation standards for efficacy, phyto-toxicity, and human/animal toxicity of dsRNA-based pesticides, as well as formal review and registration procedures. Moreover, establishing a national expert pool specializing in dsRNA pesticide assessment will be essential for effective regulatory implementation.
Conclusions and Prospects in dsRNA-Based Technology
In terms of technical and biological limitations of SIGS, a critical bottleneck of SIGS is the rapid degradation of dsRNA in open-field conditions and its limited cellular uptake, as described previously. Environmental factors such as UV radiation, rainfall, and microbial nuclease activity accelerate degradation, while internal insect factors such as nucleases in salivary glands, midgut, and hemolymph further reduce RNAi efficiency (Christiaens et al., 2020; Mitter et al., 2017; San Miguel and Scott, 2016). Since the antiviral effect of dsRNA typically lasts only a few days, frequent reapplication is required, which increases labor and cost (Dalakouras et al., 2018; Dalakouras et al., 2024; Niehl et al., 2018). The production of dsRNA remains expensive, with in vitro synthesis costing several hundred dollars per gram and in vivo microbial fermentation requiring complex optimization (Taning et al., 2016; Zotti et al., 2018). Formulation strategies such as BioClay (LDH nanoclays) and virus-like particles (VLPs) improve stability and prolong persistence, but further advances are needed for cost-effective commercialization (Jain et al., 2022; Mitter et al., 2017). One of the important issues for the establishment of SIGS is the efficacy and delivery challenges of dsRNA to target species in intercellular spaces of plants or target viruses in plant cells. Even with stabilization, SIGS efficiency depends on sufficient absorption and systemic transport of dsRNA within the plant. While chewing insects can efficiently ingest surface-applied dsRNA, sap-sucking pests rely heavily on vascular uptake, which is often inefficient. These limitations restrict the antiviral and insecticidal spectrum of SIGS (Rank and Koch, 2021). Regarding safety and ecological considerations for SIGS, nanocarriers such as carbon dots, chitosan nanoparticles, and SWNTs improve dsRNA stability and uptake but raise biosafety concerns, including potential effects on crop growth, soil biodiversity, non-target organisms, and human health (Lai, 2015; Li et al., 2015; Luo et al., 2024; Vasquez-Gutierrez et al., 2025; Wang et al., 2014). For instance, silver nanoparticles reduced germination rates in rice seedlings, whereas carbon dots disrupted microbial communities and decreased nitrogen turnover efficiency (Cheon et al., 2017; Luo et al., 2024; Thuesombat et al., 2014). Given these reports, comprehensive ecological risk assessments are required for nanomaterial-based RNA delivery technologies (Luo et al., 2024; Pérez-de-Luque, 2017). Beyond technical hurdles, SIGS technology faces regulatory ambiguity and issues of public acceptance comparable to those of GMOs (James, 2020). Concerns about off-target effects, including epigenetic modifications are important in many areas (Mamta and Rajam, 2017). Although in vivo and in silico evaluations, such as those conducted for Bayer’s SmartStax Pro corn, confirmed minimal risk of off-target effects (Bachman et al., 2020; Head et al., 2017), standardized regulatory frameworks are lacking in many regions (Voloudakis et al., 2015). Public outreach, transparent biosafety evaluations, and harmonized regulations will be critical for adoption. Despite these challenges, RNAi-based technologies remain among the most promising alternatives to conventional pesticides. By optimizing delivery systems, improving formulation stability, reducing production costs, and establishing robust regulatory frameworks, SIGS can be transformed into a practical and sustainable crop protection tool. Integration with complementary biotechnologies, including precision breeding and digital agriculture, could accelerate the evolution of SIGS into a comprehensive plant health management strategy (Mitter et al., 2017; Pérez-de-Luque, 2017). Continued investment in research, regulatory alignment, and scalable production will be pivotal for transitioning SIGS into commercial agriculture, ultimately contributing to sustainable crop production and global food security. Future integration of SIGS with digital agriculture systems, such as real-time pathogen surveillance, predictive epidemiological modeling, and precision delivery platforms, has the potential to position dsRNA-based approaches as a transformative pillar of sustainable plant health management (Mitter et al., 2017).
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